Regulation of cardiomyocyte proliferation by the Reptin ATPase

Reptin ATP酶对心肌细胞增殖的调节

• 批准号: 10747229
• 负责人: Felicia E Wranitz
• 金额: $ 3.56万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10747229
• 关键词: ATAC-seq; ATP phosphohydrolase; Adult; Birth; Bromodeoxyuridine; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cause of Death; Cell Cycle; Cell Cycle Arrest; Cell division; Chromatin; Cicatrix; Complex; Congenital Heart Defects; DNA; DNA Damage; DNA Repair; DNA-dependent ATPase; Data; Data Set; Development; Dissection; Dominant-Negative Mutation; Down-Regulation; Embryo; Engineering; Failure; Family member; Feedback; Fertilization; Gamma-H2AX; Gene Expression; Gene Expression Regulation; Genes; Genetic Models; Genetic Transcription; Heart; Heart Diseases; Heart Injuries; Human; Hyperplasia; Infarction; Injury; Learning; Life; Mediating; Messenger RNA; Modeling; Molecular; Morbidity - disease rate; Mutation; Myocardial; Myocardial Infarction; Myocardium; Natural regeneration; PRC1 Protein; Pathway interactions; Phenotype; Proliferating; Publishing; Regulation; Reporting; Repression; Resistance; Role; S-Phase Fraction; Siblings; Signal Transduction; Source; TP53 gene; Testing; Therapeutic; Transcript; Transcription Factor AP-1; Transgenes; Up-Regulation; Zebrafish; cardiac regeneration; cardiac repair; cardiogenesis; chromatin remodeling; cohort; comparison control; congenital heart disorder; cyclin A2; experimental study; healing; heart damage; insight; loss of function; loss of function mutation; mortality; mutant; novel; novel therapeutics; overexpression; postnatal; regenerative; regenerative therapy; transcriptome sequencing

ABSTRACT: Cardiomyocyte proliferation is an important source of new myocardium during heart development and regeneration. After birth cardiomyocytes are highly resistant to proliferation. The lack of adult cardiomyocyte proliferation precludes cardiac repair and underlies the high morbidity and mortality rates associated with congenital heart defects and adult cardiovascular disease. Therefore, identifying novel regulators of cardiomyocyte proliferation is key to the development of regenerative heart therapies. Zebrafish are a well- established model to study cardiac development and regeneration because their cardiomyocytes maintain a remarkable capacity to proliferate allowing the heart to regenerate after adult injury. Our lab recently reported that the AAA+ ATPase Reptin is a potent suppressor of cardiomyocyte proliferation. Reptin is a known component of the Tip60 and INO80 complexes which have roles in DNA damage repair and chromatin remodeling. Our lab demonstrated in zebrafish that reptin loss of function mutations cause myocardial hyperplasia at 3 days post fertilization (dpf). We showed that cardiomyocyte-specific overexpression of reptin rescues the cardiomyocyte hyperproliferation phenotype. We further demonstrated that inducible overexpression of reptin after adult cardiac injury resulted in decreased cardiomyocyte proliferation and failure to regenerate. Expanding on our published data, I propose to study the molecular mechanisms by which Reptin suppresses cardiomyocyte proliferation. Preliminary data suggests that the ATPase function of Reptin is essential to dampen proliferation at 3 dpf. RNA sequencing of reptin mutant and control cardiomyocytes at 3 dpf revealed the unanticipated result that the hyperproliferative reptin mutant cardiomyocytes upregulate both pro-proliferative (e.g. fosl1a & junba) and anti-proliferative (e.g. tp53 & cbx7a) transcripts. Analysis at 4 and 5 dpf revealed that the reptin mutant cardiomyocytes lose their hyperproliferative phenotype by 4 dpf and have significantly reduced proliferation rates compared to controls at 5 dpf. I hypothesize: 1) that reptin mutant cardiomyocytes require AP-1 activity for their initial proliferative burst where DNA damage accumulates and triggers tp53 upregulation to halt cell division, and 2) that compensatory upregulation of the PRC1 complex component cbx7a drives an anti-proliferative chromatin landscape in reptin mutant cardiomyocytes. In Aim 1, I will assess the sufficiency of AP-1 family members to drive cardiomyocyte proliferation in embryonic zebrafish, the accumulation of DNA damage in hyperproliferative reptin mutant cardiomyocytes, and the role of tp53 upregulation in DNA damage signaling and cell cycle arrest in reptin mutant hearts. In Aim 2, I will identify changes in the chromatin landscape that are associated with reptin loss of function and correlate those alterations to changes in gene expression. I will also investigate the ability of cbx7a to dampen cardiomyocyte proliferation in wildtype and reptin mutant backgrounds. The proposed studies will give insight into the regulation of cardiomyocyte proliferation and identify druggable pathways/targets for novel regenerative therapies.

摘要：心肌细胞增殖是心脏发育过程中新生心肌的重要来源。 和再生。出生后心肌细胞对增殖具有高度抵抗力。成人心肌细胞缺乏 增殖阻碍了心脏修复，并成为与相关疾病相关的高发病率和死亡率的基础 先天性心脏病和成人心血管疾病。因此，确定新的监管机构 心肌细胞增殖是再生心脏疗法发展的关键。斑马鱼是一种很好的 建立了研究心脏发育和再生的模型，因为它们的心肌细胞维持着 卓越的增殖能力使心脏在成人受伤后能够再生。我们实验室最近报道 AAA+ ATPase Reptin 是心肌细胞增殖的有效抑制剂。 Reptin是一种已知的 Tip60 和 INO80 复合物的组成部分，在 DNA 损伤修复和染色质中发挥作用 重塑。我们的实验室在斑马鱼中证明，Reptin 功能丧失突变会导致心肌 受精后 3 天增生 (dpf)。我们发现，心肌细胞特异性地过度表达 Reptin 挽救心肌细胞过度增殖表型。我们进一步证明了诱导性过度表达 成人心脏损伤后的雷普汀导致心肌细胞增殖减少和再生失败。 扩展我们已发表的数据，我建议研究 Reptin 抑制的分子机制 心肌细胞增殖。初步数据表明 Reptin 的 ATP 酶功能对于抑制 3 dpf 时的增殖。 3 dpf 时，reptin 突变体和对照心肌细胞的 RNA 测序揭示了 意想不到的结果是，过度增殖的 Reptin 突变心肌细胞上调促增殖 （例如 fosl1a 和 junba）和抗增殖（例如 tp53 和 cbx7a）转录本。 4 和 5 dpf 的分析表明 4 dpf 后，reptin 突变心肌细胞失去过度增殖表型，并且显着减少 与 5 dpf 时的对照相比，增殖率。我假设：1）reptin突变型心肌细胞需要 AP-1 的初始增殖爆发活性，其中 DNA 损伤累积并触发 tp53 上调以停止细胞分裂，2) PRC1 复合物的补偿性上调 成分cbx7a驱动reptin突变心肌细胞中的抗增殖染色质景观。在 目标 1，我将评估 AP-1 家族成员驱动胚胎心肌细胞增殖的充分性 斑马鱼，过度增殖的reptin突变心肌细胞中DNA损伤的积累，以及 tp53在reptin突变心脏中DNA损伤信号传导和细胞周期停滞中的上调。在目标 2 中，我将确定 与 Reptin 功能丧失相关的染色质景观的变化并将这些变化关联起来 基因表达变化的改变。我还将研究cbx7a抑制心肌细胞的能力 野生型和eptin突变体背景下的增殖。拟议的研究将深入了解监管 心肌细胞增殖并确定新型再生疗法的药物途径/靶点。